LC/MS (TOF) Analysis of Drugs and Their Glucuronide Metabolites in Urine on Titan™ C18 after Solid Phase Extraction (SPE) using Supel™-Select SCX, β-Glucuronidase Enzyme Digestion
Materials
SPE tube or plate
enzyme
standard
Oxazepam solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Hydromorphone solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Buprenorphine solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Hydrocodone solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Lorazepam solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®Morphine solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Norbuprenorphine solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Oxymorphone solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®(−)-11-nor-9-Carboxy-Δ9-THC solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Testosterone solution
1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®CONDITIONS
sample preparation
Solid Phase Extraction (SPE)
sample/matrix
human urine
SPE tube/cartridge
Supel-Select SCX, 60 mg/3 mL (54241-U)
extraction process
urine samples were spiked at 200 ng/mL each analyte and digested with 10kU of beta-glucuronidase enzyme at 60 °C for 120 min
condition
1 mL of 1% formic acid in acetonitrile followed by 1 mL water
sample addition
1.4 mL urine
washing
2 mL water followed by 1 mL of water:methanol (75:25)
elution
1 mL of 10% ammonium hydroxide in acetonitrile
eluate post-treatment
evaporate to dryness and reconstitute in 1 mL water
column
Titan C18, 5 cm x 2.1 mm I.D., 1.9 μm particles (577122-u)
mobile phase
[A] 5 mM ammonium formate; [B] 5 mM ammonium formate in acetonitrile:water (95:5)
gradient
5 to 90% B in 2.5 min, held for 0.5 min, to 5% B in 0.1 min, held for 0.5 min
flow rate
0.3 mL/min
column temp.
35 °C
detector
ESI(+) TOF, m/z 100-1000
injection
2 μL
Description
Analysis Note
A set of compounds representing five drug classes that are routinely tested in urine were extracted from urine and analyzed using LC/MS (TOF). The entire workflow, from enzymatic digestion to hydrolyze the glucuronide metabolites to analysis using a Titan UHPLC column is presented here. Cerilliant CRMS provided reliable quantification.
Sample pretreatment: urine samples were spiked at 200 ng/mL each analyte and digested with 10kU of beta-glucuronidase enzyme at 60 °C for 120 min
Legal Information
Supel is a trademark of Sigma-Aldrich Co. LLC
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